The experiments described in this proposal are directed toward a better understanding of the mechanisms that mediate splicing of mRNA precursors in eukaryotic cells. This central process of eukaryotic gene expression, and the means whereby it can be regulated, remain poorly understood. The majority of the experiments proposed concern the purification and characterization of the components of the splicing machinery. Soluble extracts of HeLa cells that initiate transcription faithfully will be screened for the ability to splice perfectly the products of transcription of adenoviral early or cellular genes by a series of increasingly stringent assays. So too will "cytoplasmic" extracts that restore splicing activity to nuclei that lost this ability during isolation. In parallel to these experiments, methods for the purification of hnRNP fractions that contained unspliced hnRNA will be developed. Such hnRNP substrates will then be used in subsequent attempts to purify components of the splicing machinery from crude extracts. Such separation of the splicing per se from synthesis of its substrate is an essential prerequisite of a feasible purification attempt. Experiments to investigate the relationship of RNA packaging in hnRNP to the sequence of a discrete hnRNA precursor and thus to processing signals and to investigate the effects of adenovirus infection upon the population of small nuclear RNA species, postulated to play roles in, for example, splicing and transport, are also described.